Prevention of graft rejection and malignant transformation would greatly enhance the safety and applicability of human embryonic (hES) and induced pluripotent (iPS) stem cell derived therapies for a broad range of degenerative diseases including cancer. The first malignancy shown to be propagated by a primitive population of cancer stem cells (CSCs) was acute myeloid leukemia (AML). Despite wide-spread implementation of targeted therapies and allogeneic hematopoietic cell transplantation (HCT), relapse related mortality rates in AML remain high (Sakamaki et al). Thus, recent clinical trials have focused on harnessing the capacity of activated NK cells to eliminate residual CSCs in AML (Rubnitz et al; North et al). In an ongoing Phase l clinical trial, NK cells from haploidentical donors were activated ex vivo with a leukemia cell line (CTV-1) membrane lysate, and infused into patients. To date, 6 AML patients received tumor activated NK (TANK) cell therapy. All have achieved complete remission (CR) for up to a year or more. The most durable CRs were associated with myelosuppression. Durable anti-leukemic responses suggest that TANKs target CSCs and protracted myelosuppression in responders indicates that TANKs may also transiently suppress hematopoietic stem cells (HSCs) and/or mesenchymal stem cells (MSCs). Indeed, research performed by ourselves and others illustrates that human MSCs are highly sensitive to IL-2 activated NK cell cytolysis and are killed more readily than differentiated cells. Notably, MSCs can be protected from NK-mediated lysis by activation of survival signaling through NF-kB and STAT3.
To enhance both the efficiency and safety of hESC or iPSC derived cellular therapies for degenerative diseases including leukemia, we aim to determine if:
1) CNDO102-activated NK cells prevent undifferentiated hESC or iPS cell engraftment and teratoma formation
2) CNDO102-activated NK cells reduce LSC engraftment
3) CNDO102-activated NK cells induced transient host HSC and/or MSC inhibition may be used as a novel, less toxic conditioning regimen to prevent rejection of normal allogeneic HSC and may be modulated by NF-kB activation.
We anticipate that these studies will inform the development of therapeutic strategies that enhance graft tolerance while eliminating malignant stem cells – a facet unique to NK and stem cell biology.
Statement of Benefit to California:
Although much is known about the genetic and epigenetic events involved in CSC production in a Acute Myeloid Leukemia (AML), comparatively little is known about the molecular pathogenesis. An ongoing Phase 1 trial in the UK is testing tumor-activated NK cells (TANKs) immunotherapy in AML patients and Coronado Biosciences, a San Francisco company is planning Phase 2 trials with CNDO102-activated NK cell therapy in AML patients; however, a detailed molecular pathogenetic characterization has been hampered by the paucity of stem and progenitor cells in AML derived blood and marrow samples. Because hESC have robust self-renewal capacity and can provide a potentially limitless source of tissue specific stem and progenitor cells in vitro, they represent an ideal model system for generating human stem cells. Thus, California hESC research harbors tremendous potential for understanding the utilization of NK cell immunotherapy in eradicating cancer stem cells, for example, in AML events that promote self-renewal and leukemic transformation.
CNDO102 activated NK cell mediated elimination of primitive stem cells from hESC and iPSC derived cellular products may obviate malignant transformation driving teratoma formation. Notably, an ongoing Phase 1 AML clinical trial has shown sustained remissions following TANK therapy, suggesting that primitive stem cells may be TANK targets. We will determine if leukemias stem cells (LSC) are susceptible to CNDO102 activated NK cell lysis. Finally, transient aplasia seen in patients receiving TANK therapy suggests that host HSC and/or MSC may be susceptible to TANK-mediated clearance. This may provide an alternative method for opening up stem cell niches and prevent graft rejection. Prevention of graft rejection and malignant transformation would greatly enhance the safety and applicability of stem cell derived therapies for a broad range of degenerative diseases including cancer.
The translational research performed in the context of this grant will not only speed the delivery of innovative NK cell immunotherapy therapies targeting undifferentiated primitive stem cells from hESC and iPSC derived cellular products and cancer stem cells from patients with AML, it will also help to train California’s future R&D workforce in addition to developing leaders in translational medicine. This grant will provide the personnel working on the project with a clear view of the importance of their research to regenerative medicine and cancer therapy and a better perspective on future career opportunities in California. It will also directly generate revenue through development and implementation of innovative therapies aimed at eradicating malignant stem cells that enhance the safety and applicability of stem cell therapies and reduce relapse related mortality in patients with frequently refractory malignancies such as AML.
This proposal is focused on activated natural killer (NK) cells, components of the innate immune system that play a major role in the rejection of tumors. An ongoing Phase I clinical trial has utilized NK cells activated ex vivo with a leukemia cell line membrane lysate to treat acute myeloid leukemia (AML) with promising early results. These tumor-activated NK (TANK) cells have achieved complete remission in some patients, with evidence for transient suppression of hematopoietic stem cells (HSCs) and/or mesenchymal stem cells (MSCs). This work is the basis for the applicant’s hypothesis that TANK cells target HSCs and MSCs and may provide a tool for enhancing graft tolerance while eliminating malignant stem cells or teratoma forming undifferentiated ESCs. The applicant proposes three Specific Aims: (1) to determine the mechanism of NK activation by a leukemia cell lysate and test whether TANK cells can kill undifferentiated human embryonic stem cells (hESCs) in vitro and in immunodeficient mice; (2) to determine whether TANK cells can target AML stem cells and prevent engraftment in an immunodeficient mouse model; and (3) to test whether TANK cells can be utilized as a less toxic conditioning regimen and target host HSCs and/or MSCs to improve the engraftment of allogeneic HSCs in an immunodeficient mouse model.
Reviewers thought the concept of using TANK cells to target undifferentiated hESCs novel but, overall, did not find this proposal to be particularly innovative, as TANK cell therapy is already in human clinical trials. They questioned the value of recapitulating the clinical strategy in mice and felt that some of the mechanistic studies could be investigated in AML patients during a trial. Reviewers were also concerned that the proposal is based heavily on data from a small human trial that has not yet been published. They felt that mechanistic studies would have more merit once the human data is validated. Reviewers did agree that the proposal could have a major impact on the treatment of AML and the development of conditioning regimens for allogeneic transplantation, but doubted whether the study would have a significant impact on strategies for tolerance induction in the classical sense.
The reviewers raised a number of serious concerns about the research plan, including the observation that the project is not particularly responsive to the RFA. This RFA was specifically directed toward the goal of inducing tolerance to stem cell grafts. Aim 1 of this proposal focuses on preventing undifferentiated hESCs and induced pluripotent stem cells (iPSCs) from engrafting and forming teratomas. While this is an important goal, it falls outside the scope of the RFA. Aim 2 of this proposal focuses on assessing TANK cell targeting of leukemic stem cells in a model of AML, also a worthy pursuit that is outside the scope of the RFA. With regard to the project’s scientific rationale, reviewers questioned the use of TANK cells as a conditioning regimen if they are indeed shown to inhibit MSCs. They noted that destruction of the host stromal layer could lead to prolonged aplasia, as the stromal microenvironment is not typically reconstituted from the donor during HSC transplantation. Thus, TANK cell targeting of host MSCs could potentially be detrimental to patients. Reviewers raised concerns with aspects of the experimental design and preliminary data as well. They noted that one figure demonstrated engraftment of leukemic cells into immunodeficient mice, but no followup studies were presented to confirm that the cells have the same cytogenetic abnormalities as the original leukemia. Reviewers cautioned that engraftment in these mouse models can be extremely variable and that the proposed studies may not be feasible. Finally, reviewers noted that Aim 3 proposed umbilical cord blood transplant without discussion of HLA-matching, which is highly relevant to NK cell response.
Reviewers described the assembled research team as well qualified to carry out the proposed experiments. Despite the absence of evidence of the PI’s grant support in the application, the reviewers recognized the Principal Investigator (PI) as an accomplished and productive immunologist with an excellent track record. Reviewers appreciated the contributions of the Co-Investigator, who provides expertise in the fields of cancer stem cells and xenograft transplantation models. Reviewers did raise two concerns about the research team, however. First, they noted that the ability to carry out the proposed research hinges on a collaboration with an industry partner and involves proprietary reagents, and, while a scientist from the company is on the research team, no letter of support was provided. Second, reviewers found the research team, consisting of 13 individuals, to be unusually and unnecessarily large for the proposed work.
Overall, while reviewers appreciated the clinical potential of the TANK cell strategy, they raised significant concerns about the scientific rationale and feasibility of the research plan and did not feel there was sufficient information in the application regarding the validity of the experimental approach. In addition, reviewers found this proposal to be largely nonresponsive to the RFA.